http:creativecommons'orglicensesbysa2'0

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http//creativecommons.org/licenses/by-sa/2.0/
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Sequence Similarity Searching Understanding
and UsingWeb Based BLAST
Dr. Joanne Fox joanne_at_bioinformatics.ubc.ca
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Concepts of Sequence Similarity Searching

• The premise
• The sequence itself is not informative it must
  be analyzed by comparative methods against
  existing databases to develop hypothesis
  concerning relatives and function.

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Important Terms for Sequence Similarity Searching
with very different meanings

• Similarity
• The extent to which nucleotide or protein
  sequences are related. The extent of similarity
  between two sequences can be based on percent
  sequence identity and/or conservation. In BLAST
  similarity refers to a positive matrix score.
• Identity
• The extent to which two (nucleotide or amino
  acid) sequences are invariant.
• Homology
• Similarity attributed to descent from a common
  ancestor.
• It is your responsibility as an informed
  bioinformatician to use these terms correctly A
  sequence is either homologous or not. Dont use
  with this term!

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Sequence Similarity Searching The Approach

• Sequence similarity searching involves the use of
  a set of algorithms (such as the BLAST programs)
  to compare a query sequence to all the sequences
  in a specified database.
• Comparisons are made in a pairwise fashion. Each
  comparison is given a score reflecting the degree
  of similarity between the query and the sequence
  being compared.
• The higher the score, the greater the degree of
  similarity. The similarity is measured and shown
  by aligning two sequences.

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Sequence Similarity Searching The Alignment

• Alignments can be global or local (this is
  algorithm specific)
• A global alignment is an optimal alignment that
  includes all characters from each sequence
  (clustal generates global alignments)
• A local alignment is an optimal alignment that
  includes only the most similar local region or
  regions (BLAST generates local alignments).

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QUERY sequence(s)
BLAST results
BLAST program
BLAST database
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Topics
BLAST program

• The different blast programs
• Understanding the BLAST algorithm
• Word size
• HSPs
• Understanding BLAST statistics
• The alignment score (S)
• Scoring Matrices
• Dealing with gaps in an alignment
• The expectation value (E)

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The BLAST algorithm

• The BLAST programs (Basic Local Alignment Search
  Tools) are a set of sequence comparison
  algorithms introduced in 1990 that are used to
  search sequence databases for optimal local
  alignments to a query.
• Altschul SF, Gish W, Miller W, Myers EW, Lipman
  DJ (1990) Basic local alignment search tool. J.
  Mol. Biol. 215403-410.
• Altschul SF, Madden TL, Schaeffer AA, Zhang J,
  Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST
  and PSI-BLAST a new generation of protein
  database search programs. NAR 253389-3402.

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http//www.ncbi.nlm.nih.gov/BLAST/
blastn
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Several different BLAST programs
 

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Other BLAST programs

• BLAST 2 Sequences (bl2seq)
• Aligns two sequences of your choice
• Can do different types of comparison ex. Blastx
• Gives dot-plot like output
• VecScreen
• Compares query with sequences of known cloning
  vectors
• Both very handy for sequencing!

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More BLAST programs

• BLAST against genomes
• Many available
• BLAST parameters pre-optimized
• Handy for mapping query to genome
• Search for short exact matches
• BLAST parameters pre-optimized
• Great for checking probes and primers

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MegaBLAST

• megaBLAST
• For aligning sequences which differ slightly due
  to sequencing errors etc.
• Very efficient for long query sequences
• Uses big word (k-tuple) sizes to start search
• Very fast
• Accepts batch submissions of ESTs
• Can upload files of sequences as queries
• More detailed info see megaBLAST pages

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How Does BLAST Really Work?

• The BLAST programs improved the overall speed of
  searches while retaining good sensitivity
  (important as databases continue to grow) by
  breaking the query and database sequences into
  fragments ("words"), and initially seeking
  matches between fragments.
• Word hits are then extended in either direction
  in an attempt to generate an alignment with a
  score exceeding the threshold of "S".

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Picture used with permission from Chapter 11 of
Bioinformatics A Practical Guide to the
Analysis of Genes and Proteins
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How Does BLAST Really Work?

• The BLAST programs improved the overall speed of
  searches while retaining good sensitivity
  (important as databases continue to grow) by
  breaking the query and database sequences into
  fragments ("words"), and initially seeking
  matches between fragments.
• Word hits are then extended in either direction
  in an attempt to generate an alignment with a
  score exceeding the threshold of "S".

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Picture used with permission from Chapter 11 of
Bioinformatics A Practical Guide to the
Analysis of Genes and Proteins
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Each BLAST hit generates an alignment that can
contain one or more these high scoring pairs
(HSPs)
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Where does the score (S) come from?

• The quality of each pair-wise alignment is
  represented as a score and the scores are ranked.
  
• Scoring matrices are used to calculate the score
  of the alignment base by base (DNA) or amino acid
  by amino acid (protein).
• The alignment score will be the sum of the scores
  for each position.

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Whats a scoring matrix?

• Substitution matrices are used for amino acid
  alignments. These are matrices in which each
  possible residue substitution is given a score
  reflecting the probability that it is related to
  the corresponding residue in the query.
• A unitary matrix is used for DNA pairs because
  each position can be given a score of 1 if it
  matches and a score of zero if it does not.

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PAM vs. BLOSUM scoring matrices

• BLOSUM 62 is the default matrix in BLAST 2.0.
  Though it is tailored for comparisons of
  moderately distant proteins, it performs well in
  detecting closer relationships. A search for
  distant relatives may be more sensitive with a
  different matrix.

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PAM vs BLOSUM scoring matrices

• The PAM Family
• PAM matrices are based on global alignments of
  closely related proteins.
• The PAM1 is the matrix calculated from
  comparisons of sequences with no more than 1
  divergence.
• Other PAM matrices are extrapolated from PAM1.

• The BLOSUM family
• BLOSUM matrices are based on local alignments.
• BLOSUM 62 is a matrix calculated from comparisons
  of sequences with no less than 62 divergence.
• All BLOSUM matrices are based on observed
  alignments they are not extrapolated from
  comparisons of closely related proteins.

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What happens if you have a gap in the alignment?

• A gap is a position in the alignment at which a
  letter is paired with a null
• Gap scores are negative. Since a single
  mutational event may cause the insertion or
  deletion of more than one residue, the presence
  of a gap is frequently ascribed more significance
  than the length of the gap.
• Hence the gap is penalized heavily, whereas a
  lesser penalty is assigned to each subsequent
  residue in the gap.

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What do the Score and the e-value really mean?

• The quality of the alignment is represented by
  the Score.
• Score (S)
• The score of an alignment is calculated as the
  sum of substitution and gap scores. Substitution
  scores are given by a look-up table (PAM, BLOSUM)
  whereas gap scores are assigned empirically .
• The significance of each alignment is computed as
  an E value.
• E value (E)
• Expectation value. The number of different
  alignments with scores equivalent to or better
  than S that are expected to occur in a database
  search by chance. The lower the E value, the more
  significant the score.

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Is the E-value the same P-value?

• E value (E)
• Expectation value. The number of different
  alignments with scores equivalent to or better
  than S that are expected to occur in a database
  search by chance. The lower the E value, the more
  significant the score.
• When E lt 0.01, P-values and E-value are nearly
  identical.
• So, the E-value is the number of times you expect
  to see your hit occur in the database (with as
  good as or better score) due to randomn chance
  alone.

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QUERY sequence(s)
BLAST results
BLAST program
BLAST database
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Topics
BLAST databases

• The different blast databases provided by the
  NCBI
• Protein databases
• Nucleotide databases
• Genomic databases
• Considerations for choosing a BLAST database
• Custom databases for BLAST

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BLAST protein databases available at through
blastp web interface _at_ NCBI
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BLAST nucleotide databases available at through
blastn web interface _at_ NCBI
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Considerations for choosing a BLAST database

• First consider your research question
• Are you looking for an ortholog in a particular
  species?
• BLAST against the genome of that species.
• Are you looking for additional members of a
  protein family across all species?
• BLAST against nr, if you cant find hits check
  wgs, htgs, and the trace archives.
• Are you looking to annotate genes in your species
  of interest?
• BLAST against known genes (RefSeq) and/or ESTs
  from a closely related species.

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When choosing a database for BLAST

• It is important to know your reagents.
• Changing your choice of database is changing your
  search space completely
• Database size affects the BLAST statistics
• record BLAST parameters, database choice,
  database size in your bioinformatics lab book,
  just as you would for your wet-bench experiments.
• Databases change rapidly and are updated
  frequently
• It may be necessary to repeat your analyses

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Creating Custom Databases for BLAST
UBiC FAQ
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QUERY sequence(s)
BLAST results
BLAST program
BLAST database
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Topics
BLAST results

• Choosing the right BLAST program
• Running a blastp search
• BLAST parameters and options to consider
• Viewing BLAST results
• Look at your alignments
• Using the BLAST taxonomy report

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http//www.ncbi.nlm.nih.gov/BLAST/
blastn
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http//www.ncbi.nlm.nih.gov/BLAST/
Program selection guide
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What BLAST program should I use? check the
NCBIs BLAST Program selection guide
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http//www.ncbi.nlm.nih.gov/BLAST/
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Input your query (gi231571) as FASTA, raw
sequence, or Accession/ID and choose your
database
database
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Links to more information can be found on the
BLAST page
links
links
links
links
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BLAST parameters and options to consider
conserved domains
Entrez query
E-value cutoff
Word size
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More BLAST parameters and options to consider
filtering
gap penalities
matrix
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Run your BLAST search
BLAST
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The BLAST Queue
click for more info
Note your RID
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Formatting and Retrieving your BLAST results
Results
options
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A graphical view of your BLAST results
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The BLAST hit list
Score
E-Value
GenBank
alignment
EntrezGene
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The BLAST pairwise alignments
Identity
Similarity
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Sorting BLAST results by Taxonomy
Taxonomy Report
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Tax BLAST Report
Summary hits by lineage
BLAST hits by organism
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BLAST statistics to record in your bioinformatics
labbook
Record the statistics that are found at bottom of
your BLAST results page
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Homology Some Guidelines

• Similarity can be indicative of homology
• Generally, if two sequences are significantly
  similar over entire length they are likely
  homologous
• Low complexity regions can be highly similar
  without being homologous
• Homologous sequences not always highly similar
• Suggested BLAST Cutoffs
• (source Chapter 11 Bioinformatics A Practical
  Guide to the Analysis of Genes and Proteins)
• For nucleotide based searches, one should look
  for hits with E-values of 10-6 or less and
  sequence identity of 70 or more
• For protein based searches, one should look for
  hits with E-values of 10-3 or less and sequence
  identity of 25 or more

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Advanced BLAST programs

• The NCBI BLAST pages have several advanced BLAST
  methods available
• PSI-BLAST
• PHI-BLAST
• RPS-BLAST
• All are powerful methods based on protein
  similarities

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PSI-BLAST

• Position Specific Iterated BLAST
• A cycling/iterative method
• Gives increased sensitivity for detecting
  distantly related proteins
• Can give insight into functional relationships
• Very refined statistical methods
• Fast still based on BLAST methods
• Simple to use

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How does PSI-BLAST work?

• First, a standard blastp is performed
• The highest scoring hits are used to generate a
  multiple alignment
• A Position Specific Scoring Matrix (PSSM) is
  generated from the multiple alignment.
• Highly conserved residues get high scores
• Less conserved residues get lower scores
• The PSSM describes the sequence similarity
  between your query and all significant blastp
  hits
• Another similarity search is performed, this time
  using the new PSSM instead of the standard BLOSUM
  or PAM matrices
• - This PSSM (scoring matrix) is now customized to
  find sequences that are related to your original
  query
• Steps 2-4 can be repeated until convergence
• Convergence occurs when no new sequences appear
  after iteration

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http//www.ncbi.nlm.nih.gov/BLAST/
PSI-BLAST
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Format results for PSI-BLAST with inclusion
E-value set at 0.005
PSI-BLAST
BLAST
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Contributors

• Special thanks to David Wishart, Andy Baxevanis,
  Stephanie Minnema, Sohrab Shah, and Francis
  Ouellette for their contributions to these
  materials
• You are now ready to complete the BLAST
  assignment